Hantaviruses infect human endothelial cells and cause two highly lethal human diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). We have shown that pathogenic NY-1V, ANDV and HTNV inhibit the early induction of IFN within human endothelial cells while a non-pathogenic hantavirus, PHV, fails to regulate IFN induction and is incapable of replicating within human endothelial cells. Thus IFN regulation is a primary determinant of hantavirus pathogenesis. Our findings indicate that the Gn-tails of pathogenic hantaviruses block RIG-I and TBK1 directed ISRE, NF-kB and IFN transcription. We recently demonstrated that only the pathogenic hantavirus Gn-tail binds TRAF3, a protein reportedly required for IFN induction, and prevents TRAF3-TBK1 complexes following expression or during infection. Since TRAF3 regulates NF-kB activation and is required for IRF-3 activation, our findings indicate that TRAF3-Gn-tail interactions are central to hantavirus regulation of IFN induction. TRAF3-TBK1 and TRAF3-NIK complexes are regulated by ubiquitination (Ub) and the pathogenic hantavirus Gn-tail contains a degron which directs its Ub and degradation. Changing 4 residues in the NY-1V Gn-tail degron to PHV residues abolishes degradation and IFN regulation by the Gn-protein and altering this determinant of hantavirus pathogenesis may be used to attenuate hantaviruses. Here we propose to analyze Gn-tail regulation of TRAF3-TBK1 complexes and NF-kB activation pathways which are required for IFN transcription. We will define interactions between the Gn-tail and TRAF3, the role of Ub in Gn-tail regulation of IFN responses and the composition of Gn-tail-TRAF3 complexeswhich block IFN induction. We will modify Gn-tail residues within pathogenic hantaviruses and evaluate IFN regulation and replication attenuation. As a result, the mechanism by which the Gn-tail blocks IFN induction will be defined and determinants of hantavirus pathogenesis will be identified. Specific Aims: 1) Define Gn-tail Interactions with TRAF3-TBK1 Signaling Complexes 2) Define the Role of Gn-tail Ub and Degradation in Regulating Cellular IFN Response; 3) Analyze the Regulation of Canonical and Noncanonical NF-kB Activation Pathways. RELEVANCE (See instructions):